Daniel G. McDonald

Inhibition of BTK and ITK with Ibrutinib Is Effective in the Prevention of Chronic Graft-versus-Host Disease in Mice.

Bruton’s Tyrosine Kinase (BTK) and IL-2 Inducible T-cell Kinase (ITK) are enzymes responsible for the phosphorylation and activation of downstream effectors in the B-cell receptor (BCR) signaling and T cell receptor (TCR) signaling pathways, respectively. Ibrutinib is an FDA-approved potent inhibitor of both BTK and ITK that impairs B-cell and T-cell function. CD4 T cells and B cells are essential for the induction of chronic graft-versus-host disease (cGVHD). We evaluated these targets by testing the ability of Ibrutinib to prevent or ameliorate cGVHD, which is one of the major complications for patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). We found that Ibrutinib significantly alleviated cGVHD across four different mouse models, accompanied by increased long-term survival and reduced clinical score. The clinical improvements in Ibrutinib-treated recipients were associated with decreased serum-autoantibodies, costimulatory molecule activation, B-cell proliferation, and glomerulonephritis compared to vehicle controls. Ibrutinib was also able to alleviate the clinical manifestations in acute GVHD (aGVHD), where the recipients were given grafts with or without B cells, suggesting that an inhibitory effect of Ibrutinib on T cells contributes to a reduction in both aGVHD and cGVHD pathogenesis. An effective prophylactic regimen is still lacking to both reduce the incidence and severity of human cGVHD following allo-HSCT. Our study shows that Ibrutinib is an effective prophylaxis against several mouse models of cGVHD with minimal toxicity and could be a promising strategy to combat human cGVHD clinically.

Calibration of the Gamma Knife Perfexion using TG‐21 and the solid water Leksell dosimetry phantom

PURPOSE To calibrate a Gamma Knife (GK) Perfexion using TG-21 with updated chamber-dependent values for modern microionization chambers in the new solid water Leksell dosimetry phantom. This work illustrates a calibration method using commercially available equipment, instruments, and an established dosimetry protocol that may be adopted at any GK center, thus reducing the interinstitutional variation in GK calibration. The calibration was verified by three third-party dosimetry checks. In addition, measurements of the relative output factors are presented and compared to available data and the new manufacturer-provided relative output factors yet to be released. METHODS An absolute dose calibration based on the TG-21 formalism, utilizing recently reported phantom material and chamber-dependent factors, was performed using a microionization chamber in a spherical solid water phantom. The result was compared to other calibration protocols based on TG-51. Independent verification of the machine output was conducted through M.D. Anderson Dosimetry Services (MDADS), using thermoluminescent dosimeters (TLDs) in an anthropomorphic head phantom; the Radiological Physics Center (RPC), using TLDs in the standard Elekta ABS plastic calibration phantom (gray phantom), included with the GK; and through a collaborative international calibration survey by the University of Pittsburgh Medical Center (UPMC) using alanine dosimeters, also in the gray phantom. The alanine dosimeters were read by the National Institute of Standards and Technology. Finally, Gafchromic EBT film was used to measure relative output factors and these factors were compared to values reported in the literature as well as new values announced for release by Elekta. The films were exposed in the solid water phantom using an included film insert accessory. RESULTS Compared to the TG-21 protocol in the solid water phantom, the modified and unmodified TG-51 calibrations resulted in dose rates which were 1.8% and 1.3% lower, respectively. Ratios of the doses measured by third parties to the dose reported showed excellent agreement. MDADS returned ratios of 1.00 and 0.98 for the two TLDs irradiated. The RPC returned a mean ratio of 0.98 of the dose reported and the UPMC alanine study returned a mean ratio of 1.008. Relative output factors were found to be 0.817 +/- 0.009 and 0.897 +/- 0.008 for the 4 and 8 mm collimators, respectively, which are in excellent agreement with revised Monte Carlo-derived relative output factors Elekta is expected to recommend with the next version of the GK treatment planning software (GAMMAPLAN version 10). CONCLUSIONS The TG-21 dosimetry protocol, performed in a solid water phantom in conjunction with modern dosimeters and phantom material and chamber-dependent factors, can yield an accurate dose measurement in the unique GK treatment geometry. The technique described here can be easily adopted by institutions worldwide since all equipment and instruments used are commercially available, thus reducing the existing interinstitutional variation in GK calibration techniques. Relative output factor measurements made in this same solid water phantom were used to verify the relative output factors provided by Elekta and agreed excellently with output factors expected to be released in conjunction with GAMMAPLAN version 10.

T-bet is critical for the development of acute graft-versus-host disease through controlling T cell differentiation and function.

T-bet is a master regulator for IFN-γ production and Th1 differentiation. We evaluated the roles of T-bet and IFN-γ in T cell responses in acute graft-versus-host disease (GVHD) and found that T-bet−/− T cells induced significantly less GVHD compared with wild-type or IFN-γ−/− counterparts in both MHC-mismatched and MHC-matched but minor histocompatibility Ag–mismatched models driven by CD4 T cells. T-bet−/−, but not IFN-γ−/−, CD4 T cells had a markedly reduced ability to cause tissue damage in liver and gut. This distinct outcome is reflected by the differential gene expression on donor CD4 T cells deficient for T-bet or IFN-γ. At mRNA and protein levels, we defined several T-bet–dependent molecules that may account for the impaired ability of T-bet−/− T cells to migrate into target organs and to produce Th1-related cytokines. Moreover, these molecules were independent of either endogenous IFN-γ, such as CXCR3 and programmed death-1, or systematic IFN-γ, such as NKG2D, I-Ab, and granzyme B. Although both T-bet−/− and IFN-γ−/− CD4 T cells are prone to differentiate into Th17 cells, polarized Th17 cells deficient for T-bet but not for IFN-γ had a significantly reduced ability to cause GVHD. Finally, T-bet−/− T cells had a compromised graft-versus-leukemia effect, which could be essentially reversed by neutralization of IL-17 in the recipients. We conclude that T-bet is required for Th1 differentiation and migration, as well as for optimal function of Th17 cells. Thus, targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD in the clinic.

RIP1 and RIP3 complex regulates radiation-induced programmed necrosis in glioblastoma

Radiation-induced necrosis (RN) is a relatively common side effect of radiation therapy for glioblastoma. However, the molecular mechanisms involved and the ways RN mechanisms differ from regulated cell death (apoptosis) are not well understood. Here, we compare the molecular mechanism of cell death (apoptosis or necrosis) of C6 glioma cells in both in vitro and in vivo (C6 othotopically allograft) models in response to low and high doses of X-ray radiation. Lower radiation doses were used to induce apoptosis, while high-dose levels were chosen to induce radiation necrosis. Our results demonstrate that active caspase-8 in this complex I induces apoptosis in response to low-dose radiation and inhibits necrosis by cleaving RIP1 and RI. When activation of caspase-8 was reduced at high doses of X-ray radiation, the RIP1/RIP3 necrosome complex II is formed. These complexes induce necrosis through the caspase-3-independent pathway mediated by calpain, cathepsin B/D, and apoptosis-inducing factor (AIF). AIF has a dual role in apoptosis and necrosis. At high doses, AIF promotes chromatinolysis and necrosis by interacting with histone H2AX. In addition, NF-κB, STAT-3, and HIF-1 play a crucial role in radiation-induced inflammatory responses embedded in a complex inflammatory network. Analysis of inflammatory markers in matched plasma and cerebrospinal fluid (CSF) isolated from in vivo specimens demonstrated the upregulation of chemokines and cytokines during the necrosis phase. Using RIP1/RIP3 kinase specific inhibitors (Nec-1, GSK′872), we also establish that the RIP1-RIP3 complex regulates programmed necrosis after either high-dose radiation or TNF-α-induced necrosis requires RIP1 and RIP3 kinases. Overall, our data shed new light on the relationship between RIP1/RIP3-mediated programmed necrosis and AIF-mediated caspase-independent programmed necrosis in glioblastoma

Comparison of radiation dose spillage from the Gamma Knife Perfexion with that from volumetric modulated arc radiosurgery during treatment of multiple brain metastases in a single fraction

OBJECT The objective of this study was to examine radiation dose distributions created by 2 competing radiosurgery modalities for treating multiple brain metastases: single-isocenter volumetric modulated arc radiosurgery (VMAS) and Gamma Knife Perfexion (GKP). In addition, the effectiveness of multiple radiosurgery quality metrics was evaluated and compared between these advanced treatment modalities. METHODS Seven anonymized MRI data sets, each showing 2-5 metastases, were used to create plans on each system. The GammaPlan (version 10.1) program was used for planning of GKP. A neurosurgeon contoured the volumes to be treated, and no planning target volume expansion was used. A prescription dose coverage of ≥ 99% was achieved for each tumor volume. The Philips Pinnacle (version 9.2) program was used for planning of VMAS, using the SmartArc optimization algorithm for delivery on a Varian iX linear accelerator. Contours were transferred from GammaPlan, and again no planning target volume expansion was used. Between 2 and 5 arcs with table angles of 90°-270° were used. Again, a V100% of ≥ 99% was achieved for each tumor volume. After planning, the MRI scans, tumor volumes, and dose information from each plan were exported according to the Digital Imaging and Communications in Medicine standard to the VelocityAI program for analysis. Brain dose-volume histograms (DVHs) for normal brain tissues were generated, and the volume of these tissues receiving 20%-90% of the prescription dose was tabulated. Finally, the prescription isodose to tumor volume ratio (PITV; Shaw et al., 1993), conformity index (CI; Paddick, 2000), gradient index (GI, Paddick and Lippitz, 2006), and conformity/gradient index (CGI, Wagner et al. 2003) were calculated for each plan. Both the PITV and CI have ideal values of 1, while the GI and CGI have ideal values of lowest and highest achievable, respectively. RESULTS The DVHs consistently showed that with VMAS a higher amount of normal brain tissues received each dose level than with GKP. These increases were largest for lower isodose levels, with the volumes of normal brain that received 20%-50% and 60%-90% of the prescription dose showing average increases of 403% and 227%, respectively. Prescription isodose conformality showed only minor differences between the 2 modalities. Radiosurgery quality metrics including measures of the dose gradient (GI and CGI) indicated that the GKP plan was superior in each case, with respective average GI and CGI values of 3.04 and 57.75 for GKP and of 10.22 and 10.85 for VMAS. Metrics evaluating prescription isodose conformality alone differed only slightly between the modalities. Average respective PITV and CI values were 2.13 and 0.53 for GKP and 2.27 and 0.51 for VMAS. CONCLUSIONS Stereotactic radiosurgery plans for the treatment of multiple metastases with VMAS delivered significantly more dose to the normal brain tissues than plans for GKP. Radiosurgery quality metrics including a measure of the dose gradient are better suited to providing contrast between modern radiosurgery treatment platforms.

Comparison of radiation treatment delivery for pancreatic cancer: Linac intensity‐modulated radiotherapy versus helical tomotherapy

Introduction: Intensity‐modulated radiotherapy (IMRT) has been shown to reduce dose to organs at risk (OAR) while adequately treating tumour volume. This study quantitatively compares the dosimetric differences from step‐and‐shoot IMRT compared with helical tomotherapy (HT) for pancreatic head cancer.

S-nitrosoglutathione-mediated STAT3 regulation in efficacy of radiotherapy and cisplatin therapy in head and neck squamous cell carcinoma

S-nitrosoglutathione (GSNO) is an endogenous nitric oxide (NO) carrier that plays a critical role in redox based NO signaling. Recent studies have reported that GSNO regulates the activities of STAT3 and NF-κB via S-nitrosylation dependent mechanisms. Since STAT3 and NF-κB are key transcription factors involved in tumor progression, chemoresistance, and metastasis of head and neck cancer, we investigated the effect of GSNO in cell culture and mouse xenograft models of head and neck squamous cell carcinoma (HNSCC). For the cell culture studies, three HNSCC cell lines were tested (SCC1, SCC14a and SCC22a). All three cell lines had constitutively activated (phosphorylated) STAT3 (Tyr705). GSNO treatment of these cell lines reversibly decreased the STAT3 phosphorylation in a concentration dependent manner. GSNO treatment also decreased the basal and cytokine-stimulated activation of NF-κB in SCC14a cells and reduced the basal low degree of nitrotyrosine by inhibition of inducible NO synthase (iNOS) expression. The reduced STAT3/NF-κB activity by GSNO treatment was correlated with the decreased cell proliferation and increased apoptosis of HNSCC cells. In HNSCC mouse xenograft model, the tumor growth was reduced by systemic treatment with GSNO and was further reduced when the treatment was combined with radiation and cisplatin. Accordingly, GSNO treatment also resulted in decreased levels of phosphorylated STAT3. In summary, these studies demonstrate that GSNO treatment blocks the NF-κB and STAT3 pathways which are responsible for cell survival, proliferation and that GSNO mediated mechanisms complement cispaltin and radiation therapy, and thus could potentiate the therapeutic effect in HNSCC.

Validation of a modern second‐check dosimetry system using a novel verification phantom

Abstract Purpose To evaluate the Mobius second‐check dosimetry system by comparing it to ionization‐chamber dose measurements collected in the recently released Mobius Verification Phantom™ (MVP). For reference, a comparison of these measurements to dose calculated in the primary treatment planning system (TPS), Varian Eclipse with the AcurosXB dose algorithm, is also provided. Finally, patient dose calculated in Mobius is compared directly to Eclipse to demonstrate typical expected results during clinical use of the Mobius system. Methods Seventeen anonymized intensity‐modulated clinical treatment plans were selected for analysis. Dose was recalculated on the MVP in both Eclipse and Mobius. These calculated doses were compared to doses measured using an A1SL ionization‐chamber in the MVP. Dose was measured and analyzed at two different chamber positions for each treatment plan. Mobius calculated dose was then compared directly to Eclipse using the following metrics; target mean dose, target D95%, global 3D gamma pass rate, and target gamma pass rate. Finally, these same metrics were used to analyze the first 36 intensity modulated cases, following clinical implementation of the Mobius system. Results The average difference between Mobius and measurement was 0.3 ± 1.3%. Differences ranged from −3.3 to + 2.2%. The average difference between Eclipse and measurement was −1.2 ± 0.7%. Eclipse vs. measurement differences ranged from −3.0 to −0.1%. For the 17 anonymized pre‐clinical cases, the average target mean dose difference between Mobius and Eclipse was 1.0 ± 1.1%. Average target D95% difference was ‐0.9 ± 2.0%. Average global gamma pass rate, using a criteria of 3%, 2 mm, was 94.4 ± 3.3%, and average gamma pass rate for the target volume only was 80.2 ± 12.3%. Results of the first 36 intensity‐modulated cases, post‐clinical implementation of Mobius, were similar to those seen for the 17 pre‐clinical test cases. Conclusion Mobius correctly calculated dose for each tested intensity modulated treatment plan, agreeing with measurement to within 3.5% for all cases analyzed. The dose calculation accuracy and independence of the Mobius system is sufficient to provide a rigorous second‐check of a modern TPS.

Assessment of a three-dimensional (3D) water scanning system for beam commissioning and measurements on a helical tomotherapy unit

Beam scanning data collected on the tomotherapy linear accelerator using the TomoScanner water scanning system is primarily used to verify the golden beam profiles included in all Helical TomoTherapy treatment planning systems (TOMO TPSs). The user is not allowed to modify the beam profiles/parameters for beam modeling within the TOMO TPSs. The authors report the first feasibility study using the Blue Phantom Helix (BPH) as an alternative to the TomoScanner (TS) system. This work establishes a benchmark dataset using BPH for target commissioning and quality assurance (QA), and quantifies systematic uncertainties between TS and BPH. Reproducibility of scanning with BPH was tested by three experienced physicists taking five sets of measurements over a six‐month period. BPH provides several enhancements over TS, including a 3D scanning arm, which is able to acquire necessary beam‐data with one tank setup, a universal chamber mount, and the OmniPro software, which allows online data collection and analysis. Discrepancies between BPH and TS were estimated by acquiring datasets with each tank. In addition, data measured with BPH and TS was compared to the golden TOMO TPS beam data. The total systematic uncertainty, defined as the combination of scanning system and beam modeling uncertainties, was determined through numerical analysis and tabulated. OmniPro was used for all analysis to eliminate uncertainty due to different data processing algorithms. The setup reproducibility of BPH remained within 0.5 mm/0.5%. Comparing BPH, TS, and Golden TPS for PDDs beyond maximum depth, the total systematic uncertainties were within 1.4 mm/2.1%. Between BPH and TPS golden data, maximum differences in the field width and penumbra of in‐plane profiles were within 0.8 and 1.1 mm, respectively. Furthermore, in cross‐plane profiles, the field width differences increased at depth greater than 10 cm up to 2.5 mm, and maximum penumbra uncertainties were 5.6 mm and 4.6 mm from TS scanning system and TPS modeling, respectively. Use of BPH reduced measurement time by 1–2 hrs per session. The BPH has been assessed as an efficient, reproducible, and accurate scanning system capable of providing a reliable benchmark beam data. With this data, a physicist can utilize the BPH in a clinical setting with an understanding of the scan discrepancy that may be encountered while validating the TPS or during routine machine QA. Without the flexibility of modifying the TPS and without a golden beam dataset from the vendor or a TPS model generated from data collected with the BPH, this represents the best solution for current clinical use of the BPH. PACS number: 87.56.Fc

MiR-34a modulates ionizing radiation-induced senescence in lung cancer cells

MicroRNAs (miRNAs) are a new class of gene expression regulators that have been implicated in tumorigenesis and modulation of the responses to cancer treatment including that of human non-small cell lung cancer (NSCLC). However, the role of miR-34a in ionizing radiation (IR)-induced senescence in NSCLC cells remains poorly understood. Here we report that IR-induced premature senescence correlates with upregulation of miR-34a expression in NSCLC cells. Ectopic overexpression of miR-34a by transfection with synthetic miR-34a mimics markedly enhances IR-induced senescence, whereas inhibition of miR-34a by transfection with a synthetic miR-34a inhibitor attenuates IR-induced senescence. Clonogenic assays reveal that treatment with miR-34a mimics augments IR-induced cell killing in human NSCLC cells. Mechanistically, we found that the senescence-promoting effect of miR-34a is associated with a dramatic down-regulation of c-Myc (Myc) expression, suggesting that miR-34a may promote IR-induced senescence via targeting Myc. In agreement with this suggestion, knockdown of Myc expression by RNAi recapitulates the senescence-promoting effect of miR-34a and enhances IR-induced cell killing in NSCLC cells. Collectively, these results demonstrate a previously unrecognized role for miR-34a in modulating IR-induced senescence in human NSCLC cells and suggest that pharmacological intervention of miR-34a expression may represent a new therapeutic strategy for improving the efficacy of lung cancer radiotherapy.MicroRNAs (miRNAs) are a new class of gene expression regulators that have been implicated in tumorigenesis and modulation of the responses to cancer treatment including that of human non-small cell lung cancer (NSCLC). However, the role of miR-34a in ionizing radiation (IR)-induced senescence in NSCLC cells remains poorly understood. Here we report that IR-induced premature senescence correlates with upregulation of miR-34a expression in NSCLC cells. Ectopic overexpression of miR-34a by transfection with synthetic miR-34a mimics markedly enhances IR-induced senescence, whereas inhibition of miR-34a by transfection with a synthetic miR-34a inhibitor attenuates IR-induced senescence. Clonogenic assays reveal that treatment with miR-34a mimics augments IR-induced cell killing in human NSCLC cells. Mechanistically, we found that the senescence-promoting effect of miR-34a is associated with a dramatic down-regulation of c-Myc (Myc) expression, suggesting that miR-34a may promote IR-induced senescence via targeting Myc. In agreement with this suggestion, knockdown of Myc expression by RNAi recapitulates the senescence-promoting effect of miR-34a and enhances IR-induced cell killing in NSCLC cells. Collectively, these results demonstrate a previously unrecognized role for miR-34a in modulating IR-induced senescence in human NSCLC cells and suggest that pharmacological intervention of miR-34a expression may represent a new therapeutic strategy for improving the efficacy of lung cancer radiotherapy.